U.S. patent number 5,561,071 [Application Number 08/532,542] was granted by the patent office on 1996-10-01 for dna and dna technology for the construction of networks to be used in chip construction and chip production (dna-chips).
Invention is credited to Ernesto di Mauro, Cornelis P. Hollenberg.
United States Patent |
5,561,071 |
Hollenberg , et al. |
October 1, 1996 |
DNA and DNA technology for the construction of networks to be used
in chip construction and chip production (DNA-chips)
Abstract
The invention relates to construction of specific molecular
microcircuits by the use of double and single stranded nucleic
acids and specific DNA-binding proteins.
Inventors: |
Hollenberg; Cornelis P. (D
usseldorf, DE), di Mauro; Ernesto (Rome,
IT) |
Family
ID: |
6385712 |
Appl.
No.: |
08/532,542 |
Filed: |
September 25, 1995 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
Issue Date |
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116556 |
Sep 7, 1993 |
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22615 |
Feb 19, 1993 |
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552938 |
Jul 16, 1990 |
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Foreign Application Priority Data
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Jul 24, 1989 [DE] |
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39 24 454.7 |
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Current U.S.
Class: |
438/503; 117/95;
257/E21.258; 257/E21.705; 435/810; 438/677; 438/758; 438/944;
438/945; 438/99; 536/22.1; 536/25.3 |
Current CPC
Class: |
B82Y
10/00 (20130101); G06N 3/123 (20130101); H01L
21/32 (20130101); H01L 25/50 (20130101); H01L
51/0093 (20130101); H01L 51/0595 (20130101); H01L
2924/0002 (20130101); Y10S 435/81 (20130101); Y10S
438/944 (20130101); Y10S 438/945 (20130101); H01L
2924/0002 (20130101); H01L 2924/00 (20130101) |
Current International
Class: |
G06N
3/00 (20060101); G06N 3/12 (20060101); H01L
21/02 (20060101); H01L 21/98 (20060101); H01L
21/70 (20060101); H01L 21/32 (20060101); H01L
51/30 (20060101); H01L 51/05 (20060101); H01L
051/40 () |
Field of
Search: |
;435/6,810,287,299
;536/22.1,25.3 ;437/1,15,16,35,36,38,51,54,80,225,238
;935/77,88 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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3924454 |
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Feb 1991 |
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DE |
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1472191 |
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May 1977 |
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GB |
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Other References
Kornberg DNA Synthesis W. H. Freeman & Co. San Francisco Calif
p. 75 (1974). .
Goodenough Genetics Holt Rinehardt & Winston NY NY p. 33, 35,
92-93 (1974). .
Hames et al Nucleic Acid Hybridization IRL Press Wash D.C. pp.
161-178 (1985). .
H. Kabata, et al., "Visualization of Single Molecules of RNA
Polymerase Sliding along DNA," Science, vol. 262, pp. 1561-1563
(1993). .
R. Zimmerman et al., "DNA Stretching on Functionalized Gold
Surfaces," Nucleic Acids Research, vol. 22, pp. 492-497 (1994).
.
W. Oldham, "The Fabrication of Microelectronic Circuits,"
Scientific American, vol. 237, pp. 110-128 (1977). .
J. Jahanmir, et al., "The Scanning Probe Microscope," Scanning
Microscopy, vol. 6, pp. 625-660 (1992). .
D. Eigler et al., "Positioning Single Atoms With A Scanning
Tunnelling Microscope," Nature, vol. 344, pp. 524-526 (1990). .
D. Eigler et al., "An Atomic Switch Realized With The Scanning
Tunnelling Microscope," Nature, vol. 352, pp. 600-603 (1991). .
A. Oliphant et al., "Defining the Sequence Specificity of
DNA-Binding Proteins by Selecting Binding Sites from
Random-Sequence Oligonucleotides: Analysis of Yeast GCN4 Protein,"
Molecular and Cellular Biology, 9:2944 (1989). .
C. Cantor et al., "Orientation in Electric Fields," Biophysical
Chemistry part II, 665-668 (1980). .
M. Buongiorno Nardelli et al., "Binding of Sea Urchin RNA
polymerase II on Homologous Histone Genes," Eur. J. Biochem, vol.
116, pp. 171-176 (1981). .
R. W. Davis et al., "Electron Microscope Heteroduplex Methods for
Mapping Regions of Base Sequence Homology in Nucleic Acids,"
Methods Enzymol. vol. 21, pp. 413-429 (1971). .
T. H. Koller et al., "An Electron Microscopic Method for Studying
Nucleic Acid-Protein Complexes. Visualization of RNA Polymerase
Bound to the DNA of Bacteriophages T7 and T3," Biopolymers, vol.
13, pp. 995-1009 (1974). .
M. Sawadogo et al., "RNA polymerase B(II) and General Transcription
Factors," Ann. Rev. Biochem., vol. 59, pp. 711-754 (1990). .
D. C. Schwartz et al., "New Techniques for Purifying Large DNAs and
Studying Their Properties and Packaging," Cold Spring Harbor Symp.
Quant. Biol., vol. 47, pp. 189-195 (1982). .
K. Kirkegaard et al., "The Cleavage of DNA by Type-I DNA
Topoisomerases," Cold Spring Harbor Symp. Quant. Biol., vol. 49,
pp. 411-419 (1984). .
G. Micheli et al, "An Electron Microscope Study of Chromosomal DNA
Replication in Different Eukaryotic Systems," Experimental Cell
Research, vol. 137, pp. 127-140 (1982). .
Oliphant et al. (1989), Molecular and Cellular Biology, vol. 9, No.
7, pp. 2944-2949. .
Trifonov et al., "Inherently Curved DNA and Its Structural
Elements," Unusual DNA Structures, Edited by Wells et al., Springer
Verlag, New York, pp. 173-187, 1987. .
Diekmann et al., "Unique Poly (dA) Poly (dT) B'-Conformation in
Cellular an Synthetic DNAs," Nucleic Acids Research, vol. 15, No.
15, pp. 6063-6074, 1987. .
Watson et al., "Molecular Biology of the Gene," The
Benjamin/Cummings Publishing Company, Inc., 4th Edition, pp.
231-234, 240-253, 286-288, 321, 469-474 and 519-523, 1987. .
McLean et al., "The Role of Sequence in the Stabilization of
Left-Handed DNA Helices In Vitro and In Vivo," Biochemica et
Biophysica Acta 950, pp. 234-254, 1988. .
Hurwitz et al., "The Enzymic Incorporation of Ribonucleotides Into
Poly-ribonucleotides and the Effect of DNA," Biochemical and
Biophysical Research Communications, vol. 3, No. 1, pp. 15-19, Jul.
1960. .
Brack, "DNA Electron Microscopy," Critical Reviews in Biochemistry,
vol. 10, pp. 113-131, Mar. 1981. .
Ferguson et al., "Quantitative Electron Microscopy of Nucleic
Acids," Advanced Techniques in Biological Electron Microscopy II,
Edited by J. Koehler, pp. 123-135, 1978, .
Efsratiadis et al., "Enzymatic In Vitro Synthesis of Globin Genes,"
Cell, vol. 7, pp. 279-288, 1976. .
Maniatis et al., "Molecular Cloning, A Laboratory Manual," Cold
Spring Harbor Laboratory, (1982) pp. 3-15, 286-291 and 446-447.
.
Weiss et al., "Enzymatic Breakage and Joining of Deoxyribonucleic
Acid," The Journal of Biological Chemistry, vol. 243, No. 17, pp.
4543-4555, Sep. 10, 1968. .
Sherman et al., "Laboratory Course Manual for Methods in Yeast
Genetics," Cold Spring Harbor Laboratory, pp. 91-103, 1986. .
Burke et al., "Cloning of Large Segments of Exogenous DNA into
Yeast by Means of Artificial Chromosome Vectors," Science, vol.
236, pp. 806-812, 1986. .
Dickerson, "The DNA Helix and How It Is Read," Sci. Am., vol. 249,
pp. 87-102, 1983. .
Johnson, et al., "Interactions Between DNA-Bound Repressors Govern
Regulation by the .lambda. Phage Repressor," Proceedings of the
National Academy of Science of USA, vol. 76, No. 10, pp. 5061-5065,
Oct. 1979. .
McAlear et al., "Biotechnical Electron Devices," Molecular
Electronic Devices, Marcel Dekker Inc., New York, pp. 175-178,
1983. .
Ulmer, "Biological Assembly of Molecular Ultracircuits," Molecular
Electronic Devices, Marcel Dekker Inc., New York, pp. 213-220,
1983. .
W. Fiers, "Structure and Function of RNA Bacteriophages,"
Comprehensive Virology, Chapter 3,, pp. 69 and 94-204. .
Van Holde Physical Biochemistry (1971) Prentice-Hall, Inc.
Englerwood Cliffs, N.J. pp. 202-220..
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Primary Examiner: Jones; W. Gary
Assistant Examiner: Marschel; Ardin H.
Attorney, Agent or Firm: Finnegan, Henderson, Farabow,
Garrett & Dunner
Parent Case Text
This application is a continuation of application Ser. No.
08/116,556 filed Sep. 7, 1993, now abandoned, which is a
continuation of Ser. No. 08/022,615 filed Feb. 19, 1993, now
abandoned, which is a continuation of Ser. No. 07/552,938, filed
Jul. 16, 1990, now abandoned.
Claims
We claim:
1. A method for the production of a nucleic acid chip which method
comprises:
(a) providing a substrate;
(b) depositing onto said substrate two or more sequence-specific
DNA-binding proteins in a predetermined deposition pattern having
beginning and ending points; and
(c) attaching to the predeposited proteins on the substrate by
site-specific deposition and orientation a preconstructed nucleic
acid sequence such that the beginning of said sequence attaches to
said beginning point protein and the ending of said sequence
attaches to said ending point protein to construct a network in
said chip.
2. A method according to claim 1 wherein the nucleic acid chip
produced is an electronic microcircuit.
3. A method according to claim 1 wherein the network further
comprises nucleic acid formed by DNA and RNA synthesis reactions
and hybridization to the preconstructed nucleic acid sequence.
4. A method according to claim 1 which further comprises depositing
electrically conductive substances onto the network.
5. A method according to claim 1 further comprising
(a) shadowing under low angle with a masking substance so that the
substrate stays free of shadow under and one side of the nucleic
acid sequences of the network; and
(b) depositing a metallo-organic chemical vapour using electron
conductive material such as doped gallium arsenide and doped
silicium on the track which remains free from the masking substance
under and/or beside said nucleic acid sequences of the network.
6. A method for the construction of a mask for the production of a
nucleic acid chip by a photolithographic procedure, comprising the
steps:
(a) producing a nucleic acid chip according to claim 1; and
(b) shadowing under low angle with a masking, electron dense
substance so that the substrate stays free of shadow under and
beside the nucleic acid sequences of the network.
7. A method for the construction of a mask for the production of a
nucleic acid chip by a photolithographic procedure, comprising the
steps:
(a) producing a nucleic acid chip according to claim 1; and
(b) converting the network in said chip into an electron dense
substance.
Description
BACKGROUND OF THE INVENTION
DNA is a polymeric compound which can be manipulated by different
physical and enzyme techniques such as denaturation/renaturation,
enzymatic synthesis, modification reactions and protein binding.
DNA technology (ref.8) allows the construction of self-assembling
networks at a ultramicroscopical or monomolecular scale (described
below). The nucleic acid networks can be used as masks in
photolythographic procedures currently used for the construction
and production of computer chips. The networks can be reproduced by
molding to produce replicas consisting of other materials or can be
used as a scaffold to deposit different materials such as n- doped
gallium arsenide or gallium arsenide, able to conduct electric
current. So constructed conducting elements can be used as
components of electronic chips. The self assembling properties of
nucleic acids can be also used to construct switching elements
needed for electronic chips.
SUMMARY OF THE INVENTION
The present invention involves a methodology that allows the
construction of molecular microcircuits using recombinant DNA
technology and related biochemical techniques.
Some advantages of the present invention are:
Miniaturization. The networks form as a consequence of programmed
reactions which are determined by the structure of the components
of the network, e.g., the base sequence of the nucleic adds.
Therefore, their design and production do not depend upon
photolythographic reproduction of a large-scale pre-designed
network. Thus, the size and precision limits intrinsic to commonly
used reproduction procedures are a priori by-passed by our method.
The size of the circuits is close to that of the thickness of a
single or double-stranded nucleic acid (from 10 to 20 .ANG.) and
far below the sizes obtainable at present.
Precision. This is determined by the high precision possible for
the reactions of nucleic acid biosynthesis: an average of one error
per 10.sup.9 nucleotides incorporated into a polymeric chain.
Furthermore the high specificity of base pairing ensures a high
precision of the assembly of the network components. Thus, both the
miniaturization and accuracy of the microcircuits obtainable by our
DNA chip technology are at least two orders of magnitude higher
than that of the normal photolythographic procedure.
In accordance with the purposes of the invention, as embodied and
broadly described herein, the invention is a molecular micronetwork
for the production of electronic microcircuits, comprising: double
stranded nucleic acid molecules whereby a molecular pattern of the
micronetwork is formed by specifically synthesized nucleic acid
molecules and fixation thereof to specific DNA-binding protein.
In further accordance with the purposes of the invention, as
embodied and broadly described herein, the invention is a molecular
micronetwork for the production of electronic microcircuits,
comprising: single stranded nucleic acid molecules whereby a
molecular pattern of the micronetwork is formed by specifically
synthesized nucleic acid molecules and fixation thereof to specific
DNA-binding proteins.
In further accordance with the purposes of the invention, as
embodied and broadly described herein, the invention is a molecular
micronetwork for the production of electronic microcircuits,
comprising: single stranded nucleic acid molecules whereby a
molecular pattern of the micronetwork is formed by hybridization of
nucleic acid molecules and fixation thereof to specific DNA-binding
proteins.
The advantages of the invention will be set forth in part and is in
the description as follows and in part will be obvious from the
description or may be learned by practice of the invention. The
advantages of the invention will be realized and attained by means
of the elements and combinations particularly pointed out in the
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawing, which are incorporated in and constitute
a part of the specification, illustrate several embodiments of the
invention and, together with the description, serve to explain the
principles of the invention.
FIG. 1 shows the steps in the shadowing technique to build a
microcircuit.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
We describe, in the following order, the principles (and relevant
examples) underlying the construction of micropatterns and their
use as electronic chips.
I Construction of nucleic acid networks
II The conversion of nucleic acid or nucleic add-protein networks
to an electricity-conducting network.
III The nucleic acid or nucleic add-protein networks described in I
and II can also be used for a photolithographic reproduction method
using the DNA network as a mask.
I CONSTRUCTION OF NUCLEIC ACID NETWORKS
1. Construction of initiation point (DWIP) and end point (DWEP) of
the DNA wire.
A network made up of nucleic acids consists of a DNA wire
initiation point (see sect.1a below), of an intermediate part (see
sect. 1b, 1c and 2-4 below), of a DNA wire end point (see sect. 1c
below). The complexity of the intermediate part can be programmed
and can consist of branch points, switches and multistranded DNA
regions (see sect.2 below).
a. DNA wire initiation point.
A DWIP (DNA Wire Initiation Point) is constructed by use of a DNA
double strand having a blunt end at one extremity and a sequence
specific single stranded extension at the other end such that only
one end is a substrate for DNA elongation by synthesis or
hybridization.
Outline 1
An oligonucleotide with the following sequence can be synthesized
in vitro:
3' and 5' indicate the 2 extremities of the nucleic acid strand.
The enzymatic polymerization of DNA by the enzyme DNA polymerase
(ref.7) proceeds by addition of monomers to the 3'-extremity (see
ref.1). The letters G,A,C,T are acronyms that indicate the
monomeric constituents of the DNA strand; they are nucleotide
monophosphates containing respectively a purine (guanine for G,
adenine for A) or a pyrimidine (cytosine for C,, thymine for T)
residue. In DNA polymers these compounds can base pair
specifically: G couples always with C, A with T. Therefore a
self-annealing reaction in a solution containing the appropriate
buffer (2x SSC solution, ref.8 p.447) at 20.degree. C. will produce
the molecule. ##STR1##
The left extremity of molecule (2) is the DWIP, the right extremity
is the growing point (that is the point onto which additional
hybridization or synthetic reactions can be performed in order to
elongate the chain and/or create branch points or switches.
Elongation may be obtained by hybridization of a preformed DNA
molecule or a reaction of DNA synthesis. Hybridization of nucleic
acids is a procedure that exploits the tendency of nucleic acids to
anneal to double strand structures (according to the rules
mentioned above: A with T, G with C), if the complementary order of
the nucleotides that compose the DNA sequence permits it.
One synthesizes according to the procedure mentioned above the
following molecule:
The hybridization reaction between molecules (2)+(3) will produce
molecule (4): ##STR2##
This molecule produced by synthesis and hybridization has one DWIP
(left) (defined above as "blunt end") and a branched extremity
(right). This branched extremity now provides two different growing
points that can be used for further elongation and branching of the
molecule, to produce a network (Scheme 1)). Many DNA sequences can
lead to the shown below structure. The length is variable.
##STR3##
Single strand interruptions in the DNA strands (indicated in Scheme
1 by the arrows), can be easily filled up by the reaction of the
enzyme DNA ligase (commercially available, i.e., from Bethesda
Research Laboratories, Boehringer Mannheim, etc, see refs. 8,9).
The synthesis of oligonucleotides (molecules 1 and 3) can be
performed with commercially available apparatus (i.e., from Applied
Biosystems or New Brunswick Scientific Company).
The DWIP can be fixed to a solid matrix by several techniques e.g.,
locally fixed charged molecules or sequence specific DNA binding
proteins (as bacteriophage DNA binding proteins, Adenovirus binding
protein, lac repressor or synthetic DNA binding proteins) or
covalent chemical binding.
Outline 2
A DNA molecule such as molecule (4) described in outline 1 can be
fixed by the following procedure to a matrix onto which the nucleic
acid network will be formed:
(i) Place, by the use of a micromanipulator, a microdrop of a
solution of a specific protein (i.e. lambda-protein repressor; see
below) on a hydrophobic surface like polyethylene and let it
dry.
(ii) Synthesize a molecule (5) which contains the sequence (4) and
(6) in such an arrangement that sequence 6 is located at the left
end of the self-annealed double strand structure: ##STR4## (iii)
Treat the hydrophobic surface with a solution of DNA molecule (5).
The specific binding of the DNA molecule to the protein molecule is
ensured by the use of the specific DNA-protein interaction.
Specificity of such interaction is a well-known phenomenon in
biological processes and several DNA-protein interaction systems
can be chosen, as detailed in the following paragraph.
Repressors are proteins which regulate gene expression, well
described for bacteria and bacteriophages systems (ref.11). These
proteins interact with DNA with extreme, sequence-determined
specificity. A sequence 12-20 nucleotides long is sufficient to
determine an absolutely selective DNA-protein interaction. For
instance: lambda repressor binds to the DNA sequence: ##STR5## lac
repressor binds to the DNA sequence: ##STR6## (iv) Thus the
.lambda.-repressor molecule fixed to the polyethylene surface will
bind a specific DNA molecule (5) with high affinity and stability
dissociation constant of the order of K.sub.m =10.sup.-13 M).
(v) An alternative procedure for sequence-specific fixation of
polymeric DNA molecules is based on the properties of specific
interaction of homogeneous repetitive polynucleotides, such as
with oligopeptides made of repetitions of amino adds, such as
polylysine, polythreonine, etc.
Specific interaction of oligonucleotides and oligopeptides is
common knowledge.
As described above, the DNA in the DWIP can consist of
complementary strands which bind specifically to the appropriate
protein (see above).
b. Extension of the DWIP.
The right extremity of the DNA molecule (5) is bifurcated and
offers two growing points, which can be elongated by hybridisation
of a presynthesized or naturally specific DNA strand of a given
length and/or by DNA synthesis.
c. DNA wire end point (DWEP).
Construction of a connection between two fixed points.
The DWEP is constructed in a fashion similar to the DWIP. The
extension reactions as described for the DWIP can also be applied
to the DWEP leading to a connection in between the DWIP and DWEP.
The connection can be obtained by hybridization of
sequence-specific nucleic add strands. Alternatively, the extension
of the DWIP can be designed to be connected directly to the DWEP by
specific hybridization of a defined DNA strand.
EXAMPLE 1 ##STR7##
In the above scheme (9) molecule (5) serves as DWIP: Block 1
symbolizes the DNA sequence that binds specifically the lambda
repressor;block 2 symbolizes the specific lac repressor binding
sequence; the symbols X1Y1, X2Y2, X3Y3 indicate any sequence of any
length or any composition, chosen according to the complexity
requirement of the micropattern (see below). These intermediate
sequences can be easily synthesized in vitro with state of art DNA
technology or can be prepared from DNA of biological origin (see
below). Sequence 10 is built up as sequence (5) but with an other
DNA sequence.
In order to obtain a fixed DNA pattern, the following operations
are required:
1)Synthesize a DWIP (i.e. molecule (5))
2)Bind it to a fixed lambda repressor molecule, as described
3)Synthesize a DWEP, as described, e.g., at the right extremity of
molecule (9)
4)Bind it to a fixed lac repressor molecule, as described for the
lambda repressor
5)The required intermediate series of DNA molecules above indicated
as X1Y1 and X2Y2 are annealed by standard DNA-DNA hybridization
procedures to both the DWIP and DWEP. DWIP and DWEP will be
located, on a hydrophobic surface, at a distance corresponding to
the length of the intermediate part (i.e. for an intermediate of a
linear length of 3000 nucleotides, the DWIP and DWEP are 1.mu.
apart).
2.Construction of branch points switches and multistranded regions
to be used in DNA wires.
The programming of synthesis of defined DNA sequences, joining them
by sequence specific hybridization and--if wanted--the sealing of
the single stranded interruptions in the double strands so
obtained, offers the possibility of constructing at will any shape
of network.
EXAMPLE 2
The following constructions are performed
1) a double stranded DNA molecule (10) ending with two protruding
single strand sequences ##STR8## 2) a double stranded molecule (11)
ending with two protruding single strand sequences complementary to
those of (10): ##STR9## 3) Molecules 10 and 11 are annealed which
leads to a double stranded loop (12) ##STR10##
Both molecules (10) and (11) can be fixed to a matrix as described
for DWIP and DWEP. The length and sequence of each branch can be
varied at will. The resulting electronic properties (see below) can
therefore be fixed in a preprogrammed fashion. One or both branches
can contain specific binding sites for proteins. The binding of the
protein allows a change in the electronic properties of the
resulting network. Protein-DNA binding systems can be used which
only bind under certain electronic conditions in the DNA strands
thereby enabling the function of a switching element.
3. Defined DNA length or amount.
DNA is available in defined amounts, sizes, and composition, e.g.,
in the form of plasmids, viral genomes or synthetic DNA. These
units can be used for the construction of DNA elements requiring a
defined amount of DNA of a defined composition. A unit bound at a
specific point determined by the DNA sequence can give desired
properties as, e.g., a contact point.
4. DNA-protein complexes.
Specific combinations of DNA sequences and DNA binding proteins can
be used to construct functional parts in a network, e.g., a pox
virus genome has a protein bound specifically at its extremity
(ref.1). This protein can be used to bind the terminal DNA fragment
at a matrix. Furthermore many regulatory proteins with specific
binding properties such as lac-repressor, .lambda.-repressor etc.
are known. Alternatively, polypeptides can be synthesized to bind
at specific DNA sequences. In addition modified nucleotides
reacting with specific antibodies can be positioned at the end of a
DNA molecule, i.e., DNA sequences that form left-handed DNA and
react with specific antibodies (ref.2).
Specific polypeptide--DNA complexes can be used to fix DNA
fragments, e.g., to a matrix or to other DNA molecules. In addition
or alternatively, antibodies can be used to stick DNA- protein
complexes to other compounds or surfaces. DNA- protein complexes
can also be used to change local electric conductance
properties.
5. Use of RNA.
Sequence-specific RNA can be synthesized in vitro on programmed DNA
templates (ref.3). The properties of RNA differ from those of DNA.
Additionally, RNA can assume, by intrastrand hybridization, any
designed secondary structure, such as hairpin-like structures
(ref.4), thus offering additional possibilities of modulation of
electric conductivity. Mixed RNA-DNA networks can be easily
obtained by programming the order of the hybridization (or
synthesis) reactions used to construct the connections between DWIP
and DWEP.
6. Further examples
EXAMPLE 3
Simplified protocol for the physical orientation of a DNA double
strand to be used as a mold, scaffold or a mask for construction of
chips:
Step 1: Construct a DWIP with a micromanipulator on a hydrophobic
surface such as polyethylene by using a micro- drop of a
.lambda.-repressor solution and letting it dry.
Step 2: Construct a DWEP as in step 1, 50 micrometer apart from the
DWIP, using an E. coli lac-repressor solution.
Step 3: Prepare a plasmid DNA molecule (ref.8) carrying both the
lac operator and the .lambda.-operator.
Since both operators can be integrated at any desired distance
within a plasmid; DNA molecules of the desired length carrying
terminal operators can be produced by using standard recombinant
DNA techniques. Using bacteriophage T4 DNA the size of the bridge
molecule could can be as long as 165 kb, whereas a small artificial
plasmid that can be amplified in E. coli could be as short as 1 kb.
Larger molecules can also be replicated and prepared in the yeast
Saccharomyces cerevisiae as minichromosomes (ref.13).
Step 4: Treat the hydrophobic surface with a solution containing
this DNA. One DNA molecule will bind selectively and directionally
to DWIP and DWEP.
EXAMPLE 4
Construction of shorter bridges can use cosmid vectors. Short
description: restrict cosmid vector DNA. Ligate with DNA of about
49 kb (approx. 15 .mu.m) which contains at one extremity a lac
operator and at the other end a .lambda.-operator. The construction
is obtained by standard genetic engineering procedures (ref.8):
Packaging the ligated DNA in vitro, transformation of E. coli,
normal selection and ampli fication procedures (ref.8). Use this
DNA in the scheme described for example 3, starting from step 3.
Distance from DWIP and DWEP=15 micrometer.
EXAMPLE 5
Larger bridges between DWIP and DWEP may be constructed by using E.
coli chromosomal DNA with specifically inserted lysogenic phage DNA
or by recombination inserted DNA segments. Larger defined DNA
segments can also be constructed and produced in the yeast
Saccharomyces cerevisiae by the use of plasmids (ref.12) or
artificial chromosomes (ref.13). Such DNA molecules carry both
.lambda.-operator and lac operator DNA sequences, spaced by any
desired distance within the DNA element used. Therefore, these DNA
molecules can bridge a broad spectrum of distance between DWIP and
DWEP, from few nucleotides to more than 1 mm (the length of the
linearized E. coli chromosome) or more mm (the length of a yeast
chromosome). Use the constructed DNA molecules as described in
example 3, starting from step 3.
II CONVERSION OF A NUCLEIC ACID OR NUCLEIC ACID-PROTEIN NETWORK TO
AN ELECTRONIC MICROCIRCUIT
The DNA networks can be used as molds or scaffolds to produce
replicas consisting of other materials. The replicas can be made as
MOSFETS (metal oxide semiconductor field effect transistors),
MESFETS (metal semiconductor FETS), and MODFETS (modulation FETS)
by depositing in various orders different materials in selected
sequences:
A) Use of shadowing technique to deposit the conductor. The
building principle is based on the construction of a molecular
nucleic acid network (as described in I) on a support of substrate
A of defined chemical characteristics allowing to perform the
following steps:
1) Shadow (low-angle) the network with substance B using techniques
currently practised for the preparation of DNA for EM (ref.5,6) but
without rotation leading to an uncovered track along the nucleic
acid (step 1 FIG. 1). The substrate is tilted by a small value
angle relative to the gas flow direction in order to obtain an
empty shadow which follows the track defined by the DNA
(refs.5,6).
2) Deposit a layer of substance C, e.g., doped gallium arsenide,
doped silicium, or a similar conductor by electrical or chemical
deposition only at the uncovered track along the nucleic acid
pattern.
3) Remove substance B and DNA leaving the conductor pattern free
step 3 of FIG. 1.
4) Deposit a second conductor D, e.g., gallium arsenide, by
metallo-organic chemical vapor deposition method (MOCVD) step 4 of
FIG. 1.
5) If desired remove substance A and replace by another support,
substance E step 5 of FIG. 1.
This procedure leads to the substitution of a molecular nucleic
acid/protein pattern by the conductor C embedded in the conductor
D.
B) Alternatively, electric or chemical deposition of the conductor
C directly onto the nucleic acid network. Continue with step 5
(step 5 of FIG. 1).
III PHOTOLITHOGRAPHIC REPRODUCTION METHOD USING THE DNA NETWORK AS
A MASK
In standard manufacturing procedures of microelectronic circuits,
large patterns are made and then photographically placed in reduced
form on the chip. In these standard procedures a circuit is
designed and used to prepare a set of final-size master masks,
which are then reproduced on chips. The DNA networks can be used
directly as master masks for the manufacture of microelectronic
circuits, avoiding size-reduction intermediate procedures, i.e.,
the DNA or DNA-protein patterns can be used directly as photomasks
in the step of the photolithographic procedure in which the
oxidised wafer (silicon dioxide or similar) coated with a layer of
a light sensitive material is exposed to ultraviolet light through
the photomask (in this case, through the DNA). Also in this case,
the network can be changed by deposition or exchange into a network
of another material as described under Section II.
REFERENCES
(1) J D Watson et al. Molecular biology of the gene, Chap. 9, p.
240-281 and refs therein. The Benjamin/Cumming Publ Comp Inc. 4th
Ed. (1987).
(2) M J McLean and R D Wells (1988) The role of sequence in the
stabilization of left-handed DNA helices in vitro and in vivo.
Biochim Biophys Acta 950, 243-254.
(3) J Hurwitz, A Bresler and R Dizingen (1960) The enzymatic
incorporation of ribonucleotides into polynucleotides and the
effect of DNA. Biochem Biophys Res Comm 3, 15-19.
(4) W Fiers (1979) Structure and function of RNA. Bacteriophages
Comp Virology 13, 69.
(5) C Brack (1981) DNA electron microscopy. CRC Critical Reviews in
Biochemistry 10, 113-169.
(6) J Ferguson and R W Davis (1978) Quantitative electron
microscopy of nucleic acids. In: Advanced Techniques in Electron
Microscopy 2, 123-171. Ed. I. Koehler, Springer, N. Y.
(7) A Efstratiadis, A M Kafatos, A M Maxam and T Maniatis (1976)
Enzymatic in vitro synthesis of globin genes. Cell 7, 279.
(8) T Maniatis, E F Fritsch and J Sambrook. Molecular Cloning, A
Laboratory Manual. Cold Spring Harbor, USA. Laboratory Press, N.Y.
(1982).
(9) B Weiss et al. (1968) Enzymatic breakage and joining of
deoxyribonucleic acid. J Biol Chem 243; 4543.
(10) A D Johnson, B I Meyer and M Ptashne (1979) Interaction
between DNA-bound repressors govern regulation by lambda phage
repressors. Proc Natl Acad Sci 76, 5061.
(11) J D Watson et al. Molecular Biology of the Gene. Chap.16 &
17. The Benjamin/Cummings Publ Com Inc., 4th Ed. (1987)
(12) F Sherman, G R Fink and J B Hicks. Laboratory Course Manual
for Methods in Yeast Genetics, Cold Spring Harbor Laboratories, USA
(1986).
(13) D T Burke, G F Carle and M V Olson (1986) Science 236,
806-812.
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